Analyzers for the determination of non-volatile substances in a liquid (e.g. ionic substances such as H+ (pH), Na+, K+, Ca++, Cl−, neutral or charged molecules such as glucose, urea or lactate) are used in medical, environmental, and industrial technology. Clinical diagnosis, in particular, relies heavily on analyzing equipment for the determination of so-called “critical care analytes” in biological fluids such as urine, plasma, serum and above all whole blood. Such systems frequently comprise diverse sensing elements for determining the respective parameters. Such sensing elements may be used for a single determination (single-use) or reused for multiple determinations (multiple-use).
Sensing elements of this kind often utilize electro-chemical sensing technologies or optical-chemical sensing technologies for the determination of gas parameters, pH-values, ionic values or metabolite values in clinical diagnostics. Preferably a plurality of sensing elements for the determination of diverse analytes are bundled into a “cartridge” (see for instance Ann. Biol. Clin. 61, 183-91, 2003).
Clinical diagnosis requires a high degree of accuracy of measurement results. In addition, a single measurement step should supply measurement values for a large number of substances. It is furthermore expected that measurement results are presented with minimum delay and that cost per measurement value is low. Often it is desirable that measurements be performed in close proximity to the patient, for instance “at the bedside”, in the physician's office or in the critical care unit.
As a consequence, time-consuming calibrating procedures involving various calibrating media prior to actual measurement will not be acceptable, especially when “single-use” sensors are concerned. Since the cost of miniaturized devices and sensing elements must be kept low, procedures requiring costly apparatus, complex sensing elements, or a plurality of fluids and other supplies are unsatisfactory.
Electrochemical sensors may be based on one of several different measurement principles, such as potentiometric, amperometric or conductometric measurement principles. All principles require the use of a reference electrode and are often applied in configurations requiring contact with a wet calibration fluid prior to measurement of the unknown sample.
U.S. Pat. No. 4,734,184 (Burleigh et al.) discloses an electrode assembly for monitoring the concentration of a number of gases and ions present in the blood. Although the assembly is stored dry to promote an extended shelf-life, the electrodes are thoroughly hydrated (wet-up) prior to use.
U.S. Pat. No. 4,654,127 (Baker et al.) discloses a sensing device equipped with species-selective sensors and a rotatable multichamber reservoir in which calibrant and sample solutions are contained but in separate chambers. A plurality of chemical species may be detected by this device. Furthermore, these commercially available sensors are stored in a high humidity package (i.e., substantially wet). This packaging method has the effect of limiting the shelf-life of these sensing devices.
U.S. Pat. No. 5,112,455 (Cozette et al.) discloses a sensing device equipped with a reference electrode and at least one substantially dry-stored sensor capable of exhibiting a response to changes in the concentration of a preselected analyte species before the sensor attains full equilibrated wet-up. However the sensor and reference electrode must contact a calibrant fluid before the electrodes attain an equilibrated “wet-up” state, to derive meaningful analytical information from such solid-state electrodes.
Optical-chemical sensors may be based on one of several different optical measurement principles, such as fluorescence, absorbance, or reflectance measurement principles. They are applied in a number of very diverse measurement configurations and, in contrast to electro-chemical sensors, optical sensors typically do not require a reference electrode or reference sensor.
An optical-chemical or optical-biochemical sensor typically consists of one or more layers of inorganic and/or organic, preferably polymeric, substances applied on a transparent carrier or substrate, with a least one layer containing a dye whose optical characteristics (absorption, luminescence) vary with the concentration of a particular analyte contained in a sample medium. Optical-biochemical sensors contain at least one biochemical or naturally-occurring biotic agent, for instance an enzyme. The carrier may be planar, cylindric, or of any other shape. For example the layers may be applied to the “wells” of micro-titration plates, at the tip of optical fibre bundles or on single optical fibres or light-guiding structures.
An optical-chemical sensor is usually able to measure reversibly and often continuously. Exceptions to this rule are certain enzyme-carrying biochemical sensors. These measure discontinuously and often consume a substrate or reactant (such as oxygen), i.e. they or their substrate or reactant are consumed or altered and must be regenerated for subsequent measurements. Since sensors generally have a limited lifetime, they must be replaced at certain intervals.
An optical-chemical sensor is placed in direct contact with the sample medium and, when exposed to light, provides optically readable information about a particular analyte of interest which is present in the sample medium (e.g. concentration, activity or partial pressure).
The majority of optical-chemical sensors require several calibration measurements with calibrating media, with the analyte concentrations being distributed over the whole measurement range. The number of calibration measurements required depends on the measurement accuracy desired in the relevant measurement range, accuracy and range varying for different applications. For example measurements of physiologic sodium levels in blood typically span at least 120 to 160 millimoles per liter and hence require calibration measurements within that range.
In order to minimize the number of calibration measurements, at least as far as the user is concerned, and to make them fast and simple, it is possible to obtain one or more of the sensor characteristics at the manufacturing site (e.g. by calibrating a production batch or lot), and to provide the relevant data together with the sensors in suitable form.
State-of-the-art devices occasionally use the term “calibration-free sensors” in their documentation. In reality there is no such thing as a calibration-free sensor. A new sensor or newly designed or developed sensor is calibrated at least once, or one or more of its characteristics are measured at least once. It is for instance possible to calibrate a production batch during sensor fabrication and subsequently to produce sensors with just this known characteristic by reproducible fabrication techniques. Furthermore it is possible to calibrate at least one sensor or a representative number of sensors of a batch and to assign the measured characteristics to all sensors of this batch. This requires sufficiently reproducible fabrication within a batch and/or reproducible fabrication of sensors between batches. It also requires reproducible fabrication of any measuring devices or instruments supplied or endorsed by the manufacture to perform the measurement. Such factory calibration is both time-consuming and expensive, requiring extremely tight control over sensor characteristics and concomitant control over the characteristics of the measuring device or instrument.
A number of solutions have been proposed in this context, for instance measuring luminescence intensity at a plurality of wavelengths, or measuring luminescence decay time of optical sensors by methods of time- or phase-resolution. As described below, very often a multiplicity of methods are applied within a single system, due to the scarcity of indicator molecules responsive to all desired analytes within their respective concentration ranges.
For example one such “calibration-free” system utilizing optical-chemical sensors is proposed for “near-patient-testing” of blood gases (PO2 and PCO2) and blood pH-value in Clin. Chim. Acta 307, 225-233, 2001. In this system the determination of PO2 is carried out by measuring the luminescence decay time of a luminescent dye immobilised on a membrane. PCO2 is determined—avoiding the use of an optical sensor—by means of the direct infrared absorption of CO2. The pH-value is determined calorimetrically (using the principle of absorbance) through multi-wavelength transmission measurement of a calorimetric pH-indicator dye immobilised on a membrane with the sample removed. Such systems employing multiple methods are often complex and expensive.
Measuring the oxygen content of a blood sample by a luminescence quenching method is also known from U.S. Pat. No. 5,564,419 (Radiometer). The method uses a luminophore whose luminescence is quenched in the presence of oxygen. The PO2 of the sample is determined by measuring the decay time of the luminescence.
In contrast to the measurement of luminescence decay time, the determination of luminescence intensity poses greater problems as regards the parameters of the components of the optical system. For sensors using luminescence indicators with long decay times (>500 nanoseconds) state-of-the-art requirements concerning the optical measurement set-up are relatively mild.
Unfortunately there are a large number of analytes, especially ions and metabolites, for which no simple indicators or indicator systems with long luminescence decay times are available. With increasing luminescence life-time of the indicator the cross-sensitivity against well-known quenching substances, especially O2, will also increase. Indicators with decay times less than 100 nanoseconds (ns) are less affected by such problems, however the accurate and calibration-free determination of such small decay times usually requires more costly and complex instrumentation. Modern medicine increasingly requests low-cost, rugged, and miniaturized analyzers which may be used in close proximity to the patient.
The determination of the pH-value of a blood sample by a colorimetric method is known from U.S. Pat. No. 5,288,646 (Radiometer), where a photometric measurement is proposed using a calorimetric (non-luminescent) pH-indicator dye which is immobilised on a membrane situated on the channel-wall of a “sampling device”. Transmission measurement using multiple analysis wavelengths is costly and requires measures to correct for variations of the characteristics of the optical components and of the light paths. Since blood absorbs light the sample must be removed from the light path prior to measurement, for instance by compressing the channel.
In the context of luminescence indicators it has been proposed (see U.S. Pat. No. 5,108,932 (Wolfbeis)) to illuminate at one wavelength, preferably at the isosbestic point, and to measure at two different wavelengths of light emission. Working with multiple wavelengths or detection at multiple wavelengths with the characteristics of the optical components fully known demands costlier technology however. In contrast to the measuring of pH-values there is a large number of analytes for which no luminescence indicators suitable for multiple wavelength methods are available.
Measuring luminescence intensity at one broad band of analytical wavelengths is particularly advantageous. In comparison with the technologies mentioned above measuring luminescence intensity has the advantage that the set-up of optical and electronic components necessary for measurement is relatively simple and may be realized with low-cost components. A disadvantage here is the fact that certain parameters of the optical components of the measurement set-up and of the individual sensors, which influence luminescence intensity, will affect the measurement result. Although it is basically possible to build optical systems and sensors with stable components and sensors whose characteristics are precisely determined, this will be unrealistic in view of the above requirements and the expense and costs involved. A solution of the problem, which is known in principle, consists in performing a single-point calibration immediately prior to the measurement in which the parameters are determined which depend on the individual measuring equipment set-up and on the individual sensor element and which influence the luminescence intensity.
According to the state of the art it is possible, for instance in the case of optical sensors based on the measurement of luminescence intensity at a broad band of analytical wavelengths, to obtain a relative characteristic (i.e. a characteristic not depending on the individual measuring system) by calibration measurements during manufacturing and to supply this characteristic, in the form of parameters (coefficients) of a mathematical equation describing the characteristic curve, together with the sensor for use in the measuring system at the user site. The parameters may be supplied in the form of bar-codes, or stored on electronic, magnetic or optical storage media. For determination of the characteristic valid in the user measurement system (i.e. the effective characteristic) at least one further measurement of luminescence intensity is required. According to the state of the art this is obtained as follows: by means of a calibration medium containing at least the analyte to be measured in known concentration, a luminescence value corresponding to this known concentration is set at the sensor of the user measurement system and luminescence is measured, giving a calibration value for the user site. The relative characteristic referenced on the calibration value at the user site will yield the effective characteristic.
For a simple optical-chemical sensor system, in which the luminescence indicator is electronically excited by irradiation with light in an absorption band and the intensity of the emitted light in an emission band is used for determination of the analyte, at least one calibration measurement performed at the user site is required.
Regarding this user-site calibration, measurement procedures and devices are known for single-use measuring elements containing one or more optical-chemical sensors and a calibration medium.
In U.S. Pat. No. 5,080,865 (Leiner) a single-use measuring element is proposed which contains one or more electrochemical or optical sensors and includes a calibration medium suitable for the given sensor(s). Prior to measurement the measuring element is inserted into the analyzer and a calibration measurement followed by the sample measurement is performed. If a liquid tonometered with one or more gases (e.g. O2 and CO2) is used, gas- and ion-sensors may be calibrated simultaneously. Storing the sensors in a liquid has the advantage that the sensors are ready for use immediately after measurement temperature has been reached. The disadvantage is that the “shelf-life” of the sensors is limited to several months when they are stored in a liquid. This is the case especially for very sensitive, enzyme carrying biosensors. A further disadvantage lies in the fact that the single-use measuring element must hold the liquid without loss during shelf-life and that a fluidic system for transport of the calibration liquid must be provided.
In U.S. Pat. No. 5,351,563 (Karpf) it is proposed to integrate a liquid storage medium (which at same time is a calibration medium for pH- and ion-sensors) into the single-use measuring element. The storage medium is displaced by a calibrating gas saturated with water vapour, following which calibration and subsequently sample measurement are performed.
U.S. Pat. No. 5,166,079 (Blackwood et al.) discloses a method and test device for competitive immunoassays using binding partners which are labelled with a fluorescent moiety. In the dry state, the reagent layer of the test device comprises an immunocomplex of an immobilized binding partner (e.g. an antibody) for the analyte (e.g. antigen) of interest and a conjugate of a labelled analyte. In practice, the label which is present in the reagent layer is optically read prior to applying the sample to the assay element. When sample liquid containing the analyte of interest has been added to the test device, the analyte present in the sample competes with the labelled analyte conjugate in the reagent layer for the available binding sites on the immobilized binding partners. The labelled analyte dissociates therefrom and is replaced by the sample analyte in a ratio appropriately equal to the relative amounts of sample analyte and labelled analyte. A second readout signal of the reagent layer is obtained when the sample has been applied which signal is inversely proportional to the amount of analyte in the sample. The ratio of the second signal to the first signal is taken and compared with that for known amounts of analyte to determine the amount of analyte in the sample. According to U.S. Pat. No. 5,166,079, the method disclosed therein allows to compensate for variations in the instrument and in the thickness of the reagent layer from element to element and yields a better precision. Importantly, the method of U.S. Pat. No. 5,166,079 is based on the displacement of fluorescent labelled analyte from the layer which is interrogated by radiation, but the analyte in the sample does not affect the fluorescent properties of the fluorescent moiety as such.
Accordingly, there remains at the present a need for a method which integrates a sensing device, preferably an optical-chemical sensor, having the requisite of long shelf life, predictable and reproducible optical response and “wet-up” characteristics, which method allows to obtain cost-effective and accurate determinations of the concentration of analytes. Such determinations are desirably made in five minutes or less, most preferably within about a minute.
Basic Principles
To enable better understanding of the present invention the relationship between the intensity S of the luminescence signal of a luminescent species A, its concentration cA and the parameters of the given measuring system will be summarized and wet calibration, known in the art, will then be described using the case of an optical sensor with an intramolecular charge transfer (ICT) dye for determination of the pH-value of a sample.
To conform with published equations concerning wet calibration the letter S was used to designate luminescence intensity. In contrast thereto, the description of the invention will use the letter L for luminescence intensity in equations and their derivation.
Parker's equation describes the relationship between luminescence intensity S of a species A and its concentration cA when excitation wavelength (ex) and emission wavelength (em) are given:S=I0kexekemεΘdcA  (a)where I0 is the intensity of the light source, kex and kem are transmission parameters of the optical components on the excitation or the emission side, and e is the sensitivity of the detector, all depending on the light wavelength λ. Photophysical parameters depending on the luminescent species are the molar absorption coefficient ε, the luminescence quantum yield Θ and the analyte concentration cA. d is the mean pathlength of light in the medium containing the species.
For a given species A the product of the parameters I0, kex, e, kem, ε, Θ and d may be combined into a new parameter kA kA=I0kexekemεΘd  (b)resulting inS=kAcA.  (c)
The properties of optical components (e.g. intensity and spectrum of the light source, spectral transmission properties of optical filters, spectral sensitivity of detectors, etc.) and of optical assemblies (length of light paths) vary within certain limits and over time. This will cause the parameter kA to have a certain variance between sensors and between devices which will also change over time (duration of operation). These variances must be taken into account when measurements requiring a high degree of accuracy and reproducibility are made. Minimizing these variances is costly and therefore economically not feasible where low-cost measuring systems are concerned.
A well-known optical-chemical sensor for pH-determination uses the ICT dye hydroxy-pyrene-trisulfonic acid (HPTA) (Ann. Biol. Clin. 61, 183-91, 2003).
The calibration curve of the sensor can be derived from the mass action law's simple relationships between pH-value and the concentration of the protonated (AH) and deprotonated (A−) dye species:pH=pK+log(cA−/cAH)  (d)
When excited near 470 nm in an aqueous environment, no luminescence at 520 nm is generated from the protonated form. The total concentration cD of the dye is the sum of the concentrations of the individual dye species:cD=cA−+cHA  (e)
At high pH-values (i.e., pH>pK+3), the protonated dye species is absent. Thus, at high pH-values cD=cA−.
Substitution of cHA in eqn. (d) by the expression cHA=cD−cA− generated from eqn. (e) and simplification yields the equation:
                                          cA            -                    cD                =                  1                      1            +                          10                              pK                -                pH                                                                        (        f        )            Eqn. (f) is equivalent to equation (g)
                                                        k              A                        ⁢                          cA              -                                                          k              A                        ⁢            cD                          =                  1                      1            +                          10                              pK                -                pH                                                                        (        g        )            and further equivalent to equation (h) in view of equation (c)
                              s                      s            m                          =                  1                      1            +                          10                              pK                -                pH                                                                        (        h        )            where S denotes the luminescence intensity at a given pH-value and Sm denotes the luminescence intensity in absence of the protonated species HA. Finally, rearrangement of eqn. (h) yields the calibration curve of the sensor (published in Ann. Biol. Clin. 61, 183-91, 2003.)
                    s        =                              s            m                                1            +                          10                              pK                -                pH                                                                        (        i        )            
The calibration curve (eqn. i) is a sigmoidal function characterized by increasing luminescence intensity in going from low to high pH-values and a point of inflection (the dye's pK-value) centred at mid-physiologic pH-values, where S is the relative luminescence intensity as a function of pH, Sm is the maximum intensity seen at high pH-values and pK is the negative log of the indicator's proton dissociation constant.
Solving equation (i) for pH gives
                    pH        =                  pK          -                      log            (                                                            S                  m                                s                            -              1                        )                                              (        j        )            from which the pH-value may be computed if the parameters S, Sm and pK are known.
To determine the pH-value the luminescence intensity S is obtained at the user site from the luminescence measurement value of the sensor in contact with the aqueous sample. The pK value is obtained by factory calibration. The value Sm at the user site is unknown and must be determined at the user site from the luminescence measurement value of the sensor in contact with an aqueous calibrating solution of known pH-value. The necessity of the determination of Sm at the user site is obvious from equation (g). The parameters kA in the numerator and denominator of the fraction are identical only if the quantities making up the parameters kA are equal. These quantities can be seen from (b). Equality will essentially hold if Sm is determined shortly before or after S is determined using one and the same measurement set-up.
Sm may for instance be determined at the user site by measuring the luminescence of the sensor in contact with an aqueous calibration medium with high pH-value.
Preferably Sm is obtained by measuring the luminescence intensity Scal of the sensor in contact with an aqueous calibration medium, whose pH-value (pHcal) is close to the pK value known from factory calibration, and by computing Sm from equation (k):Sm=Scal(1+10pK−pHcal).  (k)
U.S. Pat. No. 6,211,359 (He et al.) discloses similar characteristics for optical sensors for the determination of potassium with luminescent indicators based on the photo induced electron transfer (PET) effect. Equation 6 of U.S. Pat. No. 6,211,359 (He et al.) may also be applied in the case of other ions and in addition takes into account interfering ions which might be present. Equation 7 of U.S. Pat. No. 6,211,359 is used to obtain the concentration of the ion to be measured in analogy to eqn. (j). Eqn. 8 of U.S. Pat. No. 6,211,359 is used to find the unknown value of Sm by means of a single-point calibration in analogy to eqn. (k).
U.S. Pat. No. 6,171,866 (He et al.) discloses similar characteristics for optical sensors for the determination of calcium with luminescent indicators based on the PET effect. Eqn. 6 of U.S. Pat. No. 6,211,359 and eqn. 4 of U.S. Pat. No. 6,171,866 are equivalent with the exception that eqn. 4 does not take into account interfering ions and that the concentration and the Kd value are given in logarithmic form.
Definitions
In order to prevent misunderstandings due to varying definitions in previously published documents the following definitions are given for a number of essential concepts.
Analyte: in the following analyte will mean a substance in an aqueous sample medium to be qualitatively or quantitatively determined. The term non-volatile analyte will be used in distinction from volatile analytes, i.e. substances which are gaseous under standard conditions such as O2 or CO2. Non-volatile analytes include, e.g. ionic substances such as H+ (pH), Na+, K+, Ca++, Cl−, neutral or charged molecules such as glucose or lactate. The interaction between analyte and luminescent dye in the optical sensor can either be direct or indirect.
“Direct interaction” means that the analyte reaches the dye and both species actually react with each other.
“Indirect interaction” means that the analyte does not come into direct contact with the luminescent dye and/or that the luminescent response of the dye is not due to chemical or physical analyte-dye interaction. Examples are furnished by enzymatic sensors which belong to the group of biochemical sensors. In this context one or more enzymes react with the analyte, yielding a reaction product which in turn reacts directly with the indicator dye. In certain known biosensors the enzyme reaction causes e.g. a change in pH-value which may be determined by means of a pH-sensitive indicator dye. Examples may be found in Biosensors & Bioelectronics 10, 1995, 653-659 (Konicki et al.).
Another type of indirect interaction occurs in assays based on the fluorescence resonance energy transfer (FRET) principle (cf. infra) according to which the analyte interacts with an acceptor dye and the luminescence of a donor dye is measured.
Irrespective of whether a direct or indirect interaction of the analyte with the luminescent dye occurs, in analogy to classical pH absorption dyes these luminescent dyes are subsequently called luminescent indicator dyes.
Unless specifically mentioned, the term “analyte” in connection with its interactions with the luminescent dye shall encompass both the direct and indirect interactions as defined supra. E.g. if the non-volatile analyte is H+ and a pH-sensitive dye is used, direct interaction of the analyte and the dye occurs. If, however, glucose is the analyte and an enzyme sensor is used employing the principle of detecting a pH change which occurs when glucose is enzymatically converted, the species interacting with the dye is H+, not glucose.
In the present context, therefore, the statements like “the analyte reacts with the indicator dye”, “the analyte interacts with the indicator dye” “the analyte is bound to the dye” and similar statements shall encompass both direct and indirect analyte—dye—interactions as defined supra.
Sample medium: the sample medium typically is an aqueous solution with dissolved salts, which in addition may contain organic, biochemical or biological components. The sample media to be measured may come from the area of environmental technology (water or waste-water samples), from biotechnology and from medicine (blood, serum, plasma, urine samples or samples of other body fluids).
Optical sensor: in the usage of the present invention the term “optical sensor” refers to the interface between a sample medium and the optical components of a measuring device; in particular, it refers to one or more layers of inorganic and/or organic, preferably polymeric, substances applied on a transparent carrier or substrate, with at least one layer containing a dye whose optical characteristics (absorption, luminescence) vary with the concentration of a particular analyte contained in a sample medium. This interface is also designated as optode or optrode.
Components of the measuring system or the measuring device, such as light source, detector, optical filters, electronic signal amplifiers and the evaluation unit are not part of the optical sensor.
The present invention relates to optical sensors for the measurement of substances that are non-volatile (non-gaseous) under standard conditions, such as inorganic ions (e.g. H+, Na+, K+, Ca++, Cl−, NO3−, Fe2+, etc.), electrically neutral or charged molecules (e.g. lactate, glucose, urea, creatinine, amines, alcohols) dissolved in preferably aqueous sample media.
The present invention does not relate to optical sensors for the measurement of substances that are gaseous under standard conditions such as O2, CO2, SO2, etc. In particular, it does not relate to optical gas sensors, i.e. sensors which in the dry state and in contact with a gaseous sample medium respond to a change in the partial pressure of the analyte (e.g. O2, CO2) with a change in the optical signal. The invention does also not relate to sensors for such volatile analytes dissolved in an aqueous sample that is in contact with the sensor.
However, the present invention can be used when separate sensors for non-volatile and volatile analytes are used in combination. In this case, however, the invention is applicable only in connection with the sensors for non-volatile analytes.
Luminescence-optical sensors: the present invention preferentially relates to luminescence-optical sensors. Such sensors contain at least one luminescent dye (also referred to as luminescent indicator dye) in at least one layer.
Dry optical sensor: the term relates to an optical sensor according to the above definition, in which all sensor materials making up the sensor are dry (i.e. essentially free from water). The sensor is in this state during storage and/or prior to measurement use. To functionally activate the sensor it must be brought into contact with water or a medium containing water, for instance an aqueous activation medium, a sample medium, or a calibration medium.
Wet optical sensor: the term relates to an optical sensor according to the above definition which is in contact with an aqueous medium, for instance an aqueous activation-, sample-, or calibration-medium.
Activity: the activity a of an ionic substance is the product of its concentration c and its coefficient of activity Activity depends on ionic strength. At low ionic strength the activity coefficient is 1, and thus c=a. Depending on the application the expert will compute a suitable other value, e.g. by using the equations of Debeye-Huckel. If, in the following, the determination of the concentration is mentioned, the determination of the activity is also encompassed.
Measuring system: the term relates, with the exception of the optical sensor itself as defined above, to all optical, electronic and mechanical components which are required for application of the optical sensor, such as the light source generating the excitation radiation, the detector measuring the intensity of the measurement radiation, optical filters, electronic signal amplifiers, the evaluation unit and the measuring cell (for instance a cuvette to whose wall the sensor is attached, a cell with an inlet and possibly an outlet and a measuring passage to whose wall the sensor is attached, or a micro-titration plate).
Measuring device or device: the totality of all the components of the measuring system. Preferably, the measuring cell (containing the optical sensor) is not an integral part of the device but may be replaced together with the sensor.
(Response) characteristic or characteristic function: the characteristic describes the functional relationship between the measured intensity of the measurement radiation (e.g. the luminescence intensity) and the concentration or activity of the analyte to be determined.
In the case of optical sensors the characteristic is non-linear, i.e. the functional relationship between luminescence intensity and concentration of the analyte over the complete dynamic measurement range cannot be represented by a straight line with sufficient accuracy. Depending on the required width of the measurement range and on the required degree of accuracy it may be possible for certain applications to represent at least parts of the characteristic by straight lines.
The characteristic is determined by measuring the luminescence of the sensor for a series of aqueous calibration media with different, known concentrations of the substance to be determined, these known concentrations being distributed over the expected range of concentrations of the analyte to be determined. From these measured calibration values the characteristic is derived in the form of a table or a diagram, preferably in the form of a mathematical equation. In actual measurement the concentration of the analyte is computed using the luminescence intensity measured in contact with the sample and the characteristic function.
Effective characteristic: the characteristic valid for a given sensor together with a given measuring system. Referencing the effective characteristic obtained by a factory-site measuring system to a calibration value obtained by the factory-site measuring system results in the relative characteristic.
Relative characteristic: means a characteristic independent of the specific measuring system. The relative characteristic referenced to a calibration value obtained for a user-specific measuring system provides the effective characteristic valid for the user-specific measuring system. Typically, the relative characteristic is obtained at the factory-site (cf. also the definition for “Effective characteristic”, supra) and can be referenced to a wet or a dry calibration value (cf. also the definition for “Wet to dry relationship”, infra).
Effective and relative characteristics may be computationally transformed one into the other, provided: (a) that for the measuring system for which the effective characteristic is valid, at least one calibration value is known, (for instance the intensity of the measurement radiation of the sensor in contact with a medium of known analyte concentration); and (b) that the measuring systems used for obtaining the effective and the relative characteristic are built alike.
“Wet to dry relationship”: In the context of the present application, the “wet to dry relationship” is a relationship which allows computing at the user site the concentration of the non-volatile analyte using the user-site dry calibration value and the luminescence measurement value, both measured at the user site. The “wet to dry relationship” typically is derived from factory-site dry and wet calibration values that are obtained from measurements using a representative number of single sensors from a production batch or lot. These factory-site dry and wet calibration values then lead to the “wet to dry relationship” which is taken as a relationship which is valid for the complete production lot of which the representative sensors came from.
The “wet to dry relationship” can for example be a relative characteristic, or a relative characteristic and a ratio value, and/or the like. In connection with some typical, but not limiting, examples (cf. Examples 1, 1.1., 1.2., 1.3., 2, 2.1., and 2.2., infra) and embodiments, the following specification will show how the determination of the concentration of a non-volatile analyte can be carried out using the “wet to dry relationship”.
With reference to Example 1, in particular Examples 1.1., 1.2., and 1.3. (infra), the “wet to dry relationship” comprises a relative characteristic referenced to a wet calibration value obtained at the factory-site and a ratio value.
With reference to Example 2.1. (infra), the “wet to dry relationship” comprises a relative characteristic referenced to a dry calibration value obtained at the factory-site.
With reference to Example 2.2. (infra), the “wet to dry relationship” comprises a relative characteristic referenced to a dry calibration value based on ratio values obtained at the factory-site.
Calibration: means the determination of the characteristic. When an optical sensor is calibrated it is brought into contact with calibrating media in a measuring system, which media contain the analyte to be measured in different, known concentrations. The optically measurable response of the sensor, e.g. the luminescence intensity, referenced to the known concentration of the analyte in the calibration medium serves as a reference value for the unknown concentration of the analyte in a sample to be measured.
Prior to sample measurement the sensor may be wet or dry. If dry, it must be activated by the calibration medium. In this case the calibrating medium is also the activation medium. It is also possible to use a storage medium, if provided, as the activating and also calibrating medium. Examples for this may be found U.S. Pat. No. 5,080,865 A and in U.S. Pat. No. 5,658,451 A.
Single-point-calibration: a luminescence measurement value of the dry sensor is obtained and taken as a calibration value. From the calibration value obtained with the given measuring system and the relative characteristic obtained from a measuring system built in the same way the effective characteristic valid for the given measuring system can be derived.
Measurement and evaluation: during measurement the optical sensor is brought into contact with the sample medium, which contains the analyte in a concentration to be determined. The concentration of the analyte is found from the sensor signal measured (e.g. luminescence intensity) with reference to the effective characteristic of the optical sensor.
Factory-site calibration: the determination of the parameters of the characteristic (if eqn. 7, cited below, is used, for instance parameters Kd and q) at the factory site with the exclusive use of aqueous calibrating media is well-known and not subject of the present invention.
If some calibration steps are carried out already at the factory site using a suitable measuring system, only one calibration step (single-point-calibration) may be needed at the user site, provided a measuring system of identical design is used. A necessary condition for factory-site calibration is that the characteristic obtained at the factory site does not change until the sensor is used (or at least does not change in an unforeseeable way); changes could for instance occur during transport or during storage due to temperature effects or due to chemical or physical ageing or decomposition.
Luminescent indicator dyes: in the given context the term luminescent indicator dye, luminescent dye or luminescence-optical dye refers to all substances whose luminescent response (e.g. luminescence intensity, luminescence decay time) depends on the concentration or activity of the analyte via direct or indirect interaction.
Typically, the luminescent indicator dye is immobilized in an optical sensor, preferably in at least one sensor layer.
Depending on the type of dye or dye-system the luminescent response caused by the analyte concentration is affected by very different chemical-physical and/or photophysical mechanisms. The most important types of dyes are:                A) PET dyes        B) ICT dyes        C) FRET systems (energy transfer systems).        
As already defined supra, “direct interaction” means that the analyte reaches the dye and reacts with it.
“Indirect interaction” means that the analyte does not come into direct contact with the luminescent dye and/or that the luminescent response of the dye is not due to chemical or physical analyte-dye interaction.
PET dye: an indicator dye whose luminescence is wholly or partly quenched by photoinduced electron transfer (PET). Luminescence quenching will reduce luminescence quantum yield, luminescence intensity and luminescence decay time.
The electron transfer in a PET indicator dye takes place from an electron donor to an electronically excited electron acceptor. Donor and acceptor are covalently linked via a spacer. The spacer's function is to electronically decouple donor and acceptor. The acceptor is a luminescent substance. The donor is a receptor which is able to bind the analyte, preferably reversibly. If the bound substances are ionic substances the reactive component is also called the ionophore. In a thermodynamic equilibrium reaction the analyte reacts reversibly with the indicator dye by binding to the receptor.
From the luminescence properties (e.g. luminescence intensity, luminescence decay time) the concentration of the analyte may be inferred, for instance by evaluating the visible, or with photo-detectors measurable, intensity of the emitted light in the ultraviolet (UV), visible (VIS), or near infrared (NIR) range.
PET indicator dyes have at least one species A to which the analyte S is not bound, and at least one species B to which the analyte S is bound, the two species and the analyte being in thermodynamic equilibrium after a certain time. In the species B the PET effect is wholly or partly blocked through the binding of the analyte, which results in the luminescence intensity of B having a maximum. In the species A the PET effect is not blocked resulting in a minimum of its luminescence intensity.
Since the dye component of a PET indicator dye remains essentially unaffected by the binding of the analyte, the expert will recognize a PET indicator dye by the fact that in a given chemical environment the absorption and emission spectra of the dye of both species are essentially equal as regards spectral position. Since the total spectrum results from an addition of the spectra of the two species the binding of the analyte will change the luminescence intensity of the excitation- and emission spectrum.
Examples may be found in AP de Silva et al., Coordination Chemistry Reviews 205, 2000, 41-57 (Review of PET dyes), in He et al., Anal. Chem. 75, 2003, 549-555, FIG. 2 (PET indicator dye for Na+) and in J. Am. Chem. Soc. 125, 2003, 1468-1469, FIG. 3 PET indicator dye for K+).
ICT dye: in contrast to PET indicator dyes there is no electronic decoupling of the two parts (dye and receptor component) in an ICT dye (ICT=intramolecular charge transfer). Since the binding of the analyte substantially changes the chromophore system of the dye component, the expert will recognize ICT dyes by the fact that in a given chemical environment the absorption and emission spectra of the dye component of the two species are different as regards spectral position. Since the total spectrum results from the addition of the spectra of the two species the binding of the analyte will change the relative proportion of the two component spectra in the total spectrum.
Examples may be found in Molecular Probes, Handbook of Fluorescent Probes and Research Products, 2002, 9th ed., Ch. 21, FIG. 21.19 (SNARF-4F) and FIG. 21.24 (HPTS).
FRET dye: FRET indicator dye systems (FRET=Fluorescence Resonance Energy Transfer) essentially consist of two dyes, a luminescent donor dye and an acceptor dye. The luminescence of the donor dye is quenched by the acceptor dye via radiation-less energy transfer. Quenching of the luminescence changes luminescence intensity and luminescence decay time. The acceptor dye reacts directly or indirectly with the analyte, thus changing its absorption values (absorption spectrum) and the rate of energy transfer. From the luminescence intensity of the donor dye inferences regarding the analyte can be made. A condition among others for FRET to occur is that the absorption spectrum of at least one species of the acceptor dye overlaps at least partially with the emission spectrum of the donor dye. An advantage of FRET systems lies in the fact that the expert has a choice of many known, non-luminescent indicator dyes (especially pH-sensitive absorption dyes) and that the analyte may be determined via the more sensitive luminescence measurement. Examples may be found in U.S. Pat. No. 5,232,858 A (Wolfbeis et al.), in U.S. Pat. No. 5,942,189 A (Wolfbeis et al.) and in Anal. Chim. Acta, 1998, 364, 143-151 (Huber et al.).